PROJECT SUMMARY/ABSTRACT? PROJECT 3 Project 3's objective is to address the role of microglia in modulating transcriptional adaptations in striatal medium spiny neurons (MSNs) triggered by cocaine self-administration. Microglia contribute to normal brain development and function by supporting neuronal survival and removing non-functional neurons and synapses. We have found that ablation of microglia causes a robust increase in behavioral responses to cocaine or to a D1 dopamine receptor agonist as well as an increase in dendritic spine density on MSNs. These findings suggest the hypothesis that microglia may function homeostatically to oppose conditions of excessive dopaminergic transmission, such as seen with cocaine exposure. We also have found that 10-15% of microglia in striatum, but not other brain regions, express the D1 receptor, raising the novel possibility of direct effects of cocaine (via increased dopaminergic transmission) on microglia. We now propose to fully characterize the influence of microglia in controlling behavioral responses to cocaine in self-administration models. This will include delineating a role for all microglia in striatum as well as a possible selective role played by D1+ microglia and by D1 receptor signaling within those microglia. We will next characterize the influence of microglia?again D1+ and D1- subpopulations?on the ability of cocaine self-administration to influence gene expression profiles in the D1-type and D2-type MSNs of striatum, separately examining nucleus accumbens and dorsal striatum. We will also characterize the changes in gene expression induced by cocaine self- administration in D1+ and D1- subpopulations of microglia themselves within striatum. These experiments are made possible by several novel lines of genetic mutant mice that enable the selective manipulation of microglial subpopulations within striatum combined with RNA-seq of isolated neuronal and microglia cell types?and even at the single cell level. These investigations of cocaine set the stage for follow up studies of microglia in opiate action as well as in humans with substance use disorders. Overall, the proposal has the potential to provide paradigm-shifting information about the role of microglia in the pathophysiology of drug addiction.